High-yield isolation and subcellular proteomic characterization of nuclear and subnuclear structures from trypanosomes.
The vast evolutionary distance between the Opisthokonta (animals and yeast) and the excavata (a major group of protists, including Giardia and Trypanosoma) presents a significant challenge to in silico functional genomics and ortholog identification. Subcellular proteomic identification of the constituents of highly enriched organelles can alleviate this problem by both providing localization evidence and yielding a manageably sized proteome for detailed in silico functional assignment. We describe a method for the high-yield isolation of nuclei from the kinetoplastid Trypanosoma brucei. We also describe the subsequent purification of subnuclear compartments, including the nuclear envelope and nucleolus. Finally, using several proteomic strategies, we survey the proteome of a subcellular structure or organelle, using the nuclear pore complex as an example.
DeGrasse
Jeffrey A
JA
Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, NY, USA.
Chait
Brian T
BT
Field
Mark C
MC
Rout
Michael P
MP
D000818:Animals; D001671:Biochemistry / Q000379:methods; D002466:Cell Nucleolus / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D017886:Durapatite / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D008954:Models, Biological; D009685:Nuclear Envelope / Q000378:metabolism*; D022022:Nuclear Pore / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D013347:Subcellular Fractions; D014345:Trypanosoma / Q000378:metabolism*
D017886:Durapatite